CN104450726A - COL4A5 gene mutant and its application - Google Patents

Abstract

The invention discloses a COL4A5 gene mutant and its application, and in particular relates to separated nucleic acids encoding a COL4A5 mutant, separated peptides, a screening method of biological samples susceptible to AS syndrome, a screening system of biological samples susceptible to AS syndrome, and a kit for the screening of biological samples susceptible to AS syndrome. Compared with a SEQ ID NO:1, the separated nucleic acids encoding the COL4A5 mutant has c.1365_1373delTCCAGGCCC mutation. By detecting the existence of new mutants in a biological sample, it can effectively detect whether the biological sample is susceptible to AS syndrome.

Description

COL4A5 gene mutation body and application thereof

Technical field

The present invention relates to COL4A5 gene mutation body and application thereof.Particularly, the present invention relates to the nucleic acid being separated coding COL4A5 mutant, isolated polypeptide, the method of the syndromic biological sample of AS is easily suffered from screening, the system of the syndromic biological sample of AS is easily suffered from screening, for screening test kit, construct and the reconstitution cell of easily suffering from the syndromic biological sample of AS.

Background technology

Hereditary nephritis (i.e. Alport syndrome, in this article sometimes also referred to as " AS syndrome ") is a kind of main manifestations is blood urine, renal function Progressive symmetric erythrokeratodermia goes down, the heredity glomerular basement membrane disease of phonosensitive nerve deafness and eye exception.AS is a kind of single sick, there is genetic heterogeneity, thinks now and have 3 kinds of modes of inheritance, be i.e. sexlinked dominant inheritance, autosomal dominant inheritance and autosomal recessive inheritance.Sexlinked dominant inheritance (OMIM: 301050) be this sick central genetic mode, account for 85%, Disease-causing gene is COL4A5, and the incidence of transgenation is about 1/10000 ~ 1/5000.And 1/7 ~ 1/3 family by autosomal dominant inheritance (MIM: 203780) mode heredity, Disease-causing gene is COL4A3 and COL4A4.Autosomal recessive inheritance mode (MIM: 104200) actually rare, Disease-causing gene is autosomal gene COL4A3, and thus general consanguineous marriage just has the possibility of morbidity.The pathogenic related mechanism of this class disease has more report, but still indefinite at present.

Thus, at present the determination of the syndromic research of AS especially its pathogenic mutation is still had and treat deeply.

Summary of the invention

The present invention is intended at least to solve one of technical problem existed in prior art.For this reason, one object of the present invention is to propose a kind ofly can easily suffer from the method for the syndromic biological sample of AS by Effective selection.

The present invention completes based on the following work of contriver: contriver determines syndromic new pathogenic mutation---the c.1365_1373delTCCAGGCCC sudden change for COL4A5 gene of AS by the method that associating candidate gene sudden change checking is caught in target area.

According to a first aspect of the invention, the present invention proposes a kind of nucleic acid of coding COL4A5 mutant of separation.According to embodiments of the invention, described nucleic acid is compared with SEQ ID NO:1, have and c.1365_1373delTCCAGGCCC suddenly change, namely relative to wild-type COL4A5 gene, the cDNA of COL4A5 gene mutation body of the present invention lacks the 1365th to the 1373rd bit base TCCAGGCCC.According to embodiments of the invention, contriver determines the mutant of COL4A5 gene, this mutant and the syndromic morbidity of AS closely related, thus whether to exist in biological sample by detecting this mutant, effectively can detect biological sample and whether easily suffering from AS syndrome.

According to a second aspect of the invention, the present invention proposes a kind of isolated polypeptide.According to embodiments of the invention, compared with SEQID NO:2, p.456_458delPGP, described isolated polypeptide has suddenlys change, namely this sudden change causes owing to c.1365_1373delTCCAGGCCC suddenling change, particularly, relative to wild-type COL4A5, isolated polypeptide of the present invention (i.e. COL4A5 mutant) lacks the 456th to the 458th amino acids PGP.By detecting in biological sample whether express this polypeptide, effectively can detect biological sample and whether easily suffering from AS syndrome.

According to a third aspect of the invention we, the present invention proposes a kind of method of screening the easily syndromic biological sample of trouble AS.According to embodiments of the invention, the method comprises the following steps: from described extraction from biological material sample of nucleic acid; Determine the nucleotide sequence of described sample of nucleic acid; The nucleotide sequence of described sample of nucleic acid is compared with SEQ ID NO:1, and having c.1365_1373delTCCAGGCCC sudden change is that described biological sample easily suffers from the syndromic instruction of AS.By easily suffering from the method for the syndromic biological sample of AS according to the screening of the embodiment of the present invention, effectively can screen and easily suffer from the syndromic biological sample of AS.

According to a forth aspect of the invention, the present invention proposes a kind of system of screening the easily syndromic biological sample of trouble AS.According to embodiments of the invention, this system comprises: nucleic acid-extracting apparatus, and described nucleic acid-extracting apparatus is used for from described extraction from biological material sample of nucleic acid; Nucleotide sequence determining device, described nucleotide sequence determining device is connected with described nucleic acid-extracting apparatus, for analyzing described sample of nucleic acid, to determine the nucleotide sequence of described sample of nucleic acid; Judgment means, described judgment means is connected with described nucleotide sequence determining device, based on the nucleotide sequence of described sample of nucleic acid compared with SEQ ID NO:1, whether to have and c.1365_1373delTCCAGGCCC to suddenly change, judge whether described biological sample easily suffers from AS syndrome.Utilize this system, effectively can implement the method that the syndromic biological sample of AS is easily suffered from aforementioned screening, thus effectively can screen the easily syndromic biological sample of trouble AS.

According to a fifth aspect of the invention, the present invention proposes a kind of test kit for screening the easily syndromic biological sample of trouble AS.According to embodiments of the invention, this test kit contains: be suitable for the reagent detecting COL4A5 gene mutation body, and wherein compared with SEQ ID NO:1, this COL4A5 gene mutation body has and c.1365_1373delTCCAGGCCC suddenlys change.Utilize test kit according to an embodiment of the invention, effectively can screen and easily suffer from the syndromic biological sample of AS.

According to a sixth aspect of the invention, the invention allows for a kind of construct.According to embodiments of the invention, this construct comprises the nucleic acid of the coding COL4A5 mutant of foregoing separation.Thus, utilize the reconstitution cell that construct transformed acceptor cell of the present invention obtains, can effectively for screening the syndromic medicine for the treatment of AS.

According to a seventh aspect of the invention, the invention allows for a kind of reconstitution cell.According to embodiments of the invention, this reconstitution cell is obtained by foregoing construct transformed acceptor cell.According to some embodiments of the present invention, utilize reconstitution cell of the present invention, effectively can screen the syndromic medicine for the treatment of AS.

Additional aspect of the present invention and advantage will part provide in the following description, and part will become obvious from the following description, or be recognized by practice of the present invention.

Accompanying drawing explanation

Above-mentioned and/or additional aspect of the present invention and advantage will become obvious and easy understand from accompanying drawing below combining to the description of embodiment, wherein:

Fig. 1 shows and easily suffers from the system of the syndromic biological sample of AS and the schematic diagram of integral part thereof according to the screening of the embodiment of the present invention, wherein,

Figure 1A is the schematic diagram of the system easily suffering from the syndromic biological sample of AS according to the screening of the embodiment of the present invention,

Figure 1B is the schematic diagram of the nucleic acid-extracting apparatus according to the embodiment of the present invention,

Fig. 1 C is the schematic diagram of the nucleotide sequence determining device according to the embodiment of the present invention;

Fig. 2 shows the pedigree chart of AS syndrome patient family according to an embodiment of the invention;

Fig. 3 shows according to one embodiment of present invention, the representative Sanger sequence verification peak figure in the COL4A5 gene of non-affected males in the syndrome patient of AS shown in Fig. 2 family, female patient and male patient c.1365_1373delTCCAGGCCC mutational site.

Embodiment

Be described below in detail embodiments of the invention, the example of described embodiment is shown in the drawings, and wherein same or similar label represents same or similar element or has element that is identical or similar functions from start to finish.Being exemplary below by the embodiment be described with reference to the drawings, only for explaining the present invention, and can not limitation of the present invention being interpreted as.

COL4A5 gene mutation body

According to a first aspect of the invention, the present invention proposes a kind of nucleic acid of coding COL4A5 mutant of separation.According to embodiments of the invention, described nucleic acid, compared with SEQ ID NO:1, has and c.1365_1373delTCCAGGCCC suddenlys change.Phraseology " nucleic acid of coding COL4A5 mutant " used in this article, refer to the nucleic acid substances corresponding with the gene of COL4A5 mutant of encoding, namely the type of nucleic acid is not particularly limited, can be anyly comprise the deoxyribonucleotide corresponding with the encoding gene of COL4A5 mutant and/or the polymkeric substance of ribonucleotide, include but not limited to DNA, RNA or cDNA.According to a concrete example of the present invention, the nucleic acid of foregoing coding COL4A5 mutant is DNA.According to embodiments of the invention, contriver determines the mutant of COL4A5 gene, this mutant and the syndromic morbidity of AS closely related, thus whether exist in biological sample by detecting this mutant, effectively can detect biological sample and whether easily suffer from AS syndrome, also whether can exist in organism by detecting this mutant, effectively can predict whether organism easily suffers from AS syndrome.

For in specification sheets of the present invention and claims, mention nucleic acid, it will be appreciated by those skilled in the art that actual any of comprising complementary double-strand, or two.Conveniently, in the present specification and claims, although in most cases only give a chain, another complementary with it chain is in fact also disclosed.Such as, mention SEQ ID NO:1, actually comprise its complementary sequence.Those skilled in the art are further appreciated that and utilize a chain can detect another chain, and vice versa.

The nucleic acid of this coding COL4A5 mutant is the new pathogenic mutation of the AS syndromic Disease-causing gene COL4A5 that present inventor is determined by the method that order-checking associating candidate gene sudden change checking is caught in target area.This pathogenic mutation site is not referred in the prior art.It should be noted that, after phraseology " order-checking is caught in target area " used in this article refers to and utilizes special probe to catch enrichment to interested certain section of particular sequence of client, recycling s-generation sequencing technologies carries out the method for high-flux sequence and genome analysis.Utilize the method can obtain the genetic information of desired target area, greatly improve the Efficiency of target area in genome, significantly reduce research cost.In the present invention, the method is used for identify and study and the structure variation in the coding region of disease-related, and then, in conjunction with the data that a large amount of public databases provides, be conducive to explaining the association between gained variant structure and pathogenesis better.

Wherein, the cDNA of wild-type COL4A5 gene has nucleotide sequence as follows:

atgaaactgc gtggagtcag cctggctgcc ggcttgttct tactggccct gagtctttgg 60

gggcagcctg cagaggctgc ggcttgctat gggtgttctc caggatcaaa gtgtgactgc 120

agtggcataa aaggggaaaa gggagagaga gggtttccag gtttggaagg acacccagga 180

ttgcctggat ttccaggtcc agaagggcct ccggggcctc ggggacaaaa gggtgatgat 240

ggaattccag ggccaccagg accaaaagga atcagaggtc ctcctggact tcctggattt 300

ccagggacac caggtcttcc tggaatgcca ggccacgatg gggccccagg acctcaaggt 360

attcccggat gcaatggaac caagggagaa cgtggatttc caggcagtcc cggttttcct 420

ggtttacagg gtcctccagg accccctggg atcccaggta tgaagggtga accaggtagt 480

ataattatgt catcactgcc aggaccaaag ggtaatccag gatatccagg tcctcctgga 540

atacaaggcc tacctggtcc cactggtata ccagggccaa ttggtccccc aggaccacca 600

ggtttgatgg gccctcctgg tccaccagga cttccaggac ctaaggggaa tatgggctta 660

aatttccagg gacccaaagg tgaaaaaggt gagcaaggtc ttcagggccc acctgggcca 720

cctgggcaga tcagtgaaca gaaaagacca attgatgtag agtttcagaa aggagatcag 780

ggacttcctg gtgaccgagg gcctcctgga cctccaggga tacgtggtcc tccaggtccc 840

ccaggtggtg agaaaggtga gaagggtgag caaggagagc caggcaaaag aggtaaacca 900

ggcaaagatg gagaaaatgg ccaaccagga attcctggtt tgcctggtga tcctggttac 960

cctggtgaac ccggaaggga tggtgaaaag ggccaaaaag gtgacactgg cccacctgga 1020

cctcctggac ttgtaattcc tagacctggg actggtataa ctataggaga aaaaggaaac 1080

attgggttgc ctgggttgcc tggagaaaaa ggagagcgag gatttcctgg aatacagggt 1140

ccacctggcc ttcctggacc tccaggggct gcagttatgg gtcctcctgg ccctcctgga 1200

tttcctggag aaaggggtca gaaaggtgat gaaggaccac ctggaatttc cattcctgga 1260

cctcctggac ttgacggaca gcctggggct cctgggcttc cagggcctcc tggccctgct 1320

ggccctcaca ttcctcctag tgatgagata tgtgaaccag gccctccagg ccccccagga 1380

tctccaggtg ataaaggact ccaaggagaa caaggagtga aaggtgacaa aggtgacact 1440

tgcttcaact gcattggaac tggtatttca gggcctccag gtcaacctgg tttgccaggt 1500

ctcccaggtc ctccaggatc tcttggtttc cctggacaga aaggggaaaa aggacaagct 1560

ggtgcaactg gtcccaaagg attaccaggc attccaggag ctccaggtgc tccaggcttt 1620

cctggatcta aaggtgaacc tggtgatatc ctcacttttc caggaatgaa gggtgacaaa 1680

ggagagttgg gttcccctgg agctccaggg cttcctggtt tacctggcac tcctggacag 1740

gatggattgc cagggcttcc tggcccgaaa ggagagcctg gtggaattac ttttaagggt 1800

gaaagaggtc cccctgggaa cccaggttta ccaggcctcc cagggaatat agggcctatg 1860

ggtccccctg gtttcggccc tccaggccca gtaggtgaaa aaggcataca aggtgtggca 1920

ggaaatccag gccagccagg aataccaggt cctaaagggg atccaggtca gactataacc 1980

cagccgggga agcctggctt gcctggtaac ccaggcagag atggtgatgt aggtcttcca 2040

ggtgaccctg gacttccagg gcaaccaggc ttgccaggga tacctggtag caaaggagaa 2100

ccaggtatcc ctggaattgg gcttcctgga ccacctggtc ccaaaggctt tcctggaatt 2160

ccaggacctc caggagcacc tgggacacct ggaagaattg gtctagaagg ccctcctggg 2220

ccacccggct ttccaggacc aaagggtgaa ccaggatttg cattacctgg gccacctggg 2280

ccaccaggac ttccaggttt caaaggagca cttggtccaa aaggtgatcg tggtttccca 2340

ggacctccgg gtcctccagg acgcactggc ttagatgggc tccctggacc aaaaggtgat 2400

gttggaccaa atggacaacc tggaccaatg ggacctcctg ggctgccagg aataggtgtt 2460

cagggaccac caggaccacc agggattcct gggccaatag gtcaacctgg tttacatgga 2520

ataccaggag agaaggggga tccaggacct cctggacttg atgttccagg acccccaggt 2580

gaaagaggca gtccagggat ccccggagca cctggtccta taggacctcc aggatcacca 2640

gggcttccag gaaaagcagg tgcctctgga tttccaggta ccaaaggtga aatgggtatg 2700

atgggacctc caggcccacc aggacctttg ggaattcctg gcaggagtgg tgtacctggt 2760

cttaaaggtg atgatggctt gcagggtcag ccaggacttc ctggccctac aggagaaaaa 2820

ggtagtaaag gagagcctgg ccttccaggc cctcctggac caatggatcc aaatcttctg 2880

ggctcaaaag gagagaaggg ggaacctggc ttaccaggta tacctggagt ttcagggcca 2940

aaaggttatc agggtttgcc tggagaccca gggcaacctg gactgagtgg acaacctgga 3000

ttaccaggac caccaggtcc caaaggtaac cctggtctcc ctggacagcc aggtcttata 3060

ggacctcctg gacttaaagg aaccatcggt gatatgggtt ttccagggcc tcagggtgtg 3120

gaagggcctc ctggaccttc tggagttcct ggacaacctg gctccccagg attacctgga 3180

cagaaaggcg acaaaggtga tcctggtatt tcaagcattg gtcttccagg tcttcctggt 3240

ccaaagggtg agcctggtct gcctggatac ccagggaacc ctggtatcaa aggttctgtg 3300

ggagatcctg gtttgcccgg attaccagga acccctggag caaaaggaca accaggcctt 3360

cctggattcc caggaacccc aggccctcct ggaccaaaag gtattagtgg ccctcctggg 3420

aaccccggcc ttccaggaga acctggtcct gtaggtggtg gaggtcatcc tgggcaacca 3480

gggcctccag gcgaaaaagg caaacccggt caagatggta ttcctggacc agctggacag 3540

aagggtgaac caggtcaacc aggctttgga aacccaggac cccctggact tccaggactt 3600

tctggccaaa agggtgatgg aggattacct gggattccag gaaatcctgg ccttccaggt 3660

ccaaagggcg aaccaggctt tcacggtttc cctggtgtgc agggtccccc aggccctcct 3720

ggttctccgg gtccagctct ggaaggacct aaaggcaacc ctgggcccca aggtcctcct 3780

gggagaccag gtctaccagg tccagaaggt cctccaggtc tccctggaaa tggaggtatt 3840

aaaggagaga agggaaatcc aggccaacct gggctacctg gcttgcctgg tttgaaagga 3900

gatcaaggac caccaggact ccagggtaat cctggccggc cgggtctcaa tggaatgaaa 3960

ggagatcctg gtctccctgg tgttccagga ttcccaggca tgaaaggacc cagtggagta 4020

cctggatcag ctggccctga gggggaaccg ggacttattg gtcctccagg tcctcctgga 4080

ttacctggtc cttcaggaca gagtatcata attaaaggag atgctggtcc tccaggaatc 4140

cctggccagc ctgggctaaa gggtctacca ggaccccaag gacctcaagg cttaccaggt 4200

ccaactggcc ctccaggaga tcctggacgc aatggactcc ctggctttga tggtgcagga 4260

gggcgcaaag gagacccagg tctgccagga cagccaggta cccgtggttt ggatggtccc 4320

cctggtccag atggattgca aggtccccca ggtccccctg gaacctcctc tgttgcacat 4380

ggatttctta ttacacgcca cagccagaca acggatgcac cacaatgccc acagggaaca 4440

cttcaggtct atgaaggctt ttctctcctg tatgtacaag gaaataaaag agcccacggt 4500

caagacttgg ggacggctgg cagctgcctt cgtcgcttta gtaccatgcc tttcatgttc 4560

tgcaacatca ataatgtttg caactttgct tcaagaaatg actattctta ctggctctct 4620

accccagagc ccatgccaat gagcatgcaa cccctaaagg gccagagcat ccagccattc 4680

attagtcgat gtgcagtatg tgaagctcca gctgtggtga tcgcagttca cagtcagacg 4740

atccagattc cccattgtcc tcagggatgg gattctctgt ggattggtta ttccttcatg 4800

atgcatacaa gtgcaggggc agaaggctca ggtcaagccc tagcctcccc tggttcctgc 4860

ttggaagagt ttcgttcagc tcccttcatc gaatgtcatg ggaggggtac ctgtaactac 4920

tatgccaact cctacagctt ttggctggca actgtagatg tgtcagacat gttcagtaaa 4980

cctcagtcag aaacgctgaa agcaggagac ttgaggacac gaattagccg atgtcaagtg 5040

tgcatgaaga ggacataa 5058 (SEQ ID NO：1)，

The protein of its coding has aminoacid sequence as follows:

MKLRGVSLAA GLFLLALSLW GQPAEAAACY GCSPGSKCDC SGIKGEKGER GFPGLEGHPG 60

LPGFPGPEGP PGPRGQKGDD GIPGPPGPKG IRGPPGLPGF PGTPGLPGMP GHDGAPGPQG 120

IPGCNGTKGE RGFPGSPGFP GLQGPPGPPG IPGMKGEPGS IIMSSLPGPK GNPGYPGPPG 180

IQGLPGPTGI PGPIGPPGPP GLMGPPGPPG LPGPKGNMGL NFQGPKGEKG EQGLQGPPGP 240

PGQISEQKRP IDVEFQKGDQ GLPGDRGPPG PPGIRGPPGP PGGEKGEKGE QGEPGKRGKP 300

GKDGENGQPG IPGLPGDPGY PGEPGRDGEK GQKGDTGPPG PPGLVIPRPG TGITIGEKGN 360

IGLPGLPGEK GERGFPGIQG PPGLPGPPGA AVMGPPGPPG FPGERGQKGD EGPPGISIPG 420

PPGLDGQPGA PGLPGPPGPA GPHIPPSDEI CEPGPPGPPG SPGDKGLQGE QGVKGDKGDT 480

CFNCIGTGIS GPPGQPGLPG LPGPPGSLGF PGQKGEKGQA GATGPKGLPG IPGAPGAPGF 540

PGSKGEPGDI LTFPGMKGDK GELGSPGAPG LPGLPGTPGQ DGLPGLPGPK GEPGGITFKG 600

ERGPPGNPGL PGLPGNIGPM GPPGFGPPGP VGEKGIQGVA GNPGQPGIPG PKGDPGQTIT 660

QPGKPGLPGN PGRDGDVGLP GDPGLPGQPG LPGIPGSKGE PGIPGIGLPG PPGPKGFPGI 720

PGPPGAPGTP GRIGLEGPPG PPGFPGPKGE PGFALPGPPG PPGLPGFKGA LGPKGDRGFP 780

GPPGPPGRTG LDGLPGPKGD VGPNGQPGPM GPPGLPGIGV QGPPGPPGIP GPIGQPGLHG 840

IPGEKGDPGP PGLDVPGPPG ERGSPGIPGA PGPIGPPGSP GLPGKAGASG FPGTKGEMGM 900

MGPPGPPGPL GIPGRSGVPG LKGDDGLQGQ PGLPGPTGEK GSKGEPGLPG PPGPMDPNLL 960

GSKGEKGEPG LPGIPGVSGP KGYQGLPGDP GQPGLSGQPG LPGPPGPKGN PGLPGQPGLI 1020

GPPGLKGTIG DMGFPGPQGV EGPPGPSGVP GQPGSPGLPG QKGDKGDPGI SSIGLPGLPG 1080

PKGEPGLPGY PGNPGIKGSV GDPGLPGLPG TPGAKGQPGL PGFPGTPGPP GPKGISGPPG 1140

NPGLPGEPGP VGGGGHPGQP GPPGEKGKPG QDGIPGPAGQ KGEPGQPGFG NPGPPGLPGL 1200

SGQKGDGGLP GIPGNPGLPG PKGEPGFHGF PGVQGPPGPP GSPGPALEGP KGNPGPQGPP 1260

GRPGLPGPEG PPGLPGNGGI KGEKGNPGQP GLPGLPGLKG DQGPPGLQGN PGRPGLNGMK 1320

GDPGLPGVPG FPGMKGPSGV PGSAGPEGEP GLIGPPGPPG LPGPSGQSII IKGDAGPPGI 1380

PGQPGLKGLP GPQGPQGLPG PTGPPGDPGR NGLPGFDGAG GRKGDPGLPG QPGTRGLDGP 1440

PGPDGLQGPP GPPGTSSVAH GFLITRHSQT TDAPQCPQGT LQVYEGFSLL YVQGNKRAHG 1500

QDLGTAGSCL RRFSTMPFMF CNINNVCNFA SRNDYSYWLS TPEPMPMSMQ PLKGQSIQPF 1560

ISRCAVCEAP AVVIAVHSQT IQIPHCPQGW DSLWIGYSFM MHTSAGAEGS GQALASPGSC 1620

LEEFRSAPFI ECHGRGTCNY YANSYSFWLA TVDVSDMFSK PQSETLKAGD LRTRISRCQV 1680

CMKRT 1685 (SEQ ID NO：2)。

The cDNA sequence of COL4A5 gene mutation body of the present invention is as follows:

atgaaactgc gtggagtcag cctggctgcc ggcttgttct tactggccct gagtctttgg 60

gggcagcctg cagaggctgc ggcttgctat gggtgttctc caggatcaaa gtgtgactgc 120

agtggcataa aaggggaaaa gggagagaga gggtttccag gtttggaagg acacccagga 180

ttgcctggat ttccaggtcc agaagggcct ccggggcctc ggggacaaaa gggtgatgat 240

ggaattccag ggccaccagg accaaaagga atcagaggtc ctcctggact tcctggattt 300

ccagggacac caggtcttcc tggaatgcca ggccacgatg gggccccagg acctcaaggt 360

attcccggat gcaatggaac caagggagaa cgtggatttc caggcagtcc cggttttcct 420

ggtttacagg gtcctccagg accccctggg atcccaggta tgaagggtga accaggtagt 480

ataattatgt catcactgcc aggaccaaag ggtaatccag gatatccagg tcctcctgga 540

atacaaggcc tacctggtcc cactggtata ccagggccaa ttggtccccc aggaccacca 600

ggtttgatgg gccctcctgg tccaccagga cttccaggac ctaaggggaa tatgggctta 660

aatttccagg gacccaaagg tgaaaaaggt gagcaaggtc ttcagggccc acctgggcca 720

cctgggcaga tcagtgaaca gaaaagacca attgatgtag agtttcagaa aggagatcag 780

ggacttcctg gtgaccgagg gcctcctgga cctccaggga tacgtggtcc tccaggtccc 840

ccaggtggtg agaaaggtga gaagggtgag caaggagagc caggcaaaag aggtaaacca 900

ggcaaagatg gagaaaatgg ccaaccagga attcctggtt tgcctggtga tcctggttac 960

cctggtgaac ccggaaggga tggtgaaaag ggccaaaaag gtgacactgg cccacctgga 1020

cctcctggac ttgtaattcc tagacctggg actggtataa ctataggaga aaaaggaaac 1080

attgggttgc ctgggttgcc tggagaaaaa ggagagcgag gatttcctgg aatacagggt 1140

ccacctggcc ttcctggacc tccaggggct gcagttatgg gtcctcctgg ccctcctgga 1200

tttcctggag aaaggggtca gaaaggtgat gaaggaccac ctggaatttc cattcctgga 1260

cctcctggac ttgacggaca gcctggggct cctgggcttc cagggcctcc tggccctgct 1320

ggccctcaca ttcctcctag tgatgagata tgtgaaccag gccccccagg atctccaggt 1380

gataaaggac tccaaggaga acaaggagtg aaaggtgaca aaggtgacac ttgcttcaac 1440

tgcattggaa ctggtatttc agggcctcca ggtcaacctg gtttgccagg tctcccaggt 1500

cctccaggat ctcttggttt ccctggacag aaaggggaaa aaggacaagc tggtgcaact 1560

ggtcccaaag gattaccagg cattccagga gctccaggtg ctccaggctt tcctggatct 1620

aaaggtgaac ctggtgatat cctcactttt ccaggaatga agggtgacaa aggagagttg 1680

ggttcccctg gagctccagg gcttcctggt ttacctggca ctcctggaca ggatggattg 1740

ccagggcttc ctggcccgaa aggagagcct ggtggaatta cttttaaggg tgaaagaggt 1800

ccccctggga acccaggttt accaggcctc ccagggaata tagggcctat gggtccccct 1860

ggtttcggcc ctccaggccc agtaggtgaa aaaggcatac aaggtgtggc aggaaatcca 1920

ggccagccag gaataccagg tcctaaaggg gatccaggtc agactataac ccagccgggg 1980

aagcctggct tgcctggtaa cccaggcaga gatggtgatg taggtcttcc aggtgaccct 2040

ggacttccag ggcaaccagg cttgccaggg atacctggta gcaaaggaga accaggtatc 2100

cctggaattg ggcttcctgg accacctggt cccaaaggct ttcctggaat tccaggacct 2160

ccaggagcac ctgggacacc tggaagaatt ggtctagaag gccctcctgg gccacccggc 2220

tttccaggac caaagggtga accaggattt gcattacctg ggccacctgg gccaccagga 2280

cttccaggtt tcaaaggagc acttggtcca aaaggtgatc gtggtttccc aggacctccg 2340

ggtcctccag gacgcactgg cttagatggg ctccctggac caaaaggtga tgttggacca 2400

aatggacaac ctggaccaat gggacctcct gggctgccag gaataggtgt tcagggacca 2460

ccaggaccac cagggattcc tgggccaata ggtcaacctg gtttacatgg aataccagga 2520

gagaaggggg atccaggacc tcctggactt gatgttccag gacccccagg tgaaagaggc 2580

agtccaggga tccccggagc acctggtcct ataggacctc caggatcacc agggcttcca 2640

ggaaaagcag gtgcctctgg atttccaggt accaaaggtg aaatgggtat gatgggacct 2700

ccaggcccac caggaccttt gggaattcct ggcaggagtg gtgtacctgg tcttaaaggt 2760

gatgatggct tgcagggtca gccaggactt cctggcccta caggagaaaa aggtagtaaa 2820

ggagagcctg gccttccagg ccctcctgga ccaatggatc caaatcttct gggctcaaaa 2880

ggagagaagg gggaacctgg cttaccaggt atacctggag tttcagggcc aaaaggttat 2940

cagggtttgc ctggagaccc agggcaacct ggactgagtg gacaacctgg attaccagga 3000

ccaccaggtc ccaaaggtaa ccctggtctc cctggacagc caggtcttat aggacctcct 3060

ggacttaaag gaaccatcgg tgatatgggt tttccagggc ctcagggtgt ggaagggcct 3120

cctggacctt ctggagttcc tggacaacct ggctccccag gattacctgg acagaaaggc 3180

gacaaaggtg atcctggtat ttcaagcatt ggtcttccag gtcttcctgg tccaaagggt 3240

gagcctggtc tgcctggata cccagggaac cctggtatca aaggttctgt gggagatcct 3300

ggtttgcccg gattaccagg aacccctgga gcaaaaggac aaccaggcct tcctggattc 3360

ccaggaaccc caggccctcc tggaccaaaa ggtattagtg gccctcctgg gaaccccggc 3420

cttccaggag aacctggtcc tgtaggtggt ggaggtcatc ctgggcaacc agggcctcca 3480

ggcgaaaaag gcaaacccgg tcaagatggt attcctggac cagctggaca gaagggtgaa 3540

ccaggtcaac caggctttgg aaacccagga ccccctggac ttccaggact ttctggccaa 3600

aagggtgatg gaggattacc tgggattcca ggaaatcctg gccttccagg tccaaagggc 3660

gaaccaggct ttcacggttt ccctggtgtg cagggtcccc caggccctcc tggttctccg 3720

ggtccagctc tggaaggacc taaaggcaac cctgggcccc aaggtcctcc tgggagacca 3780

ggtctaccag gtccagaagg tcctccaggt ctccctggaa atggaggtat taaaggagag 3840

aagggaaatc caggccaacc tgggctacct ggcttgcctg gtttgaaagg agatcaagga 3900

ccaccaggac tccagggtaa tcctggccgg ccgggtctca atggaatgaa aggagatcct 3960

ggtctccctg gtgttccagg attcccaggc atgaaaggac ccagtggagt acctggatca 4020

gctggccctg agggggaacc gggacttatt ggtcctccag gtcctcctgg attacctggt 4080

ccttcaggac agagtatcat aattaaagga gatgctggtc ctccaggaat ccctggccag 4140

cctgggctaa agggtctacc aggaccccaa ggacctcaag gcttaccagg tccaactggc 4200

cctccaggag atcctggacg caatggactc cctggctttg atggtgcagg agggcgcaaa 4260

ggagacccag gtctgccagg acagccaggt acccgtggtt tggatggtcc ccctggtcca 4320

gatggattgc aaggtccccc aggtccccct ggaacctcct ctgttgcaca tggatttctt 4380

attacacgcc acagccagac aacggatgca ccacaatgcc cacagggaac acttcaggtc 4440

tatgaaggct tttctctcct gtatgtacaa ggaaataaaa gagcccacgg tcaagacttg 4500

gggacggctg gcagctgcct tcgtcgcttt agtaccatgc ctttcatgtt ctgcaacatc 4560

aataatgttt gcaactttgc ttcaagaaat gactattctt actggctctc taccccagag 4620

cccatgccaa tgagcatgca acccctaaag ggccagagca tccagccatt cattagtcga 4680

tgtgcagtat gtgaagctcc agctgtggtg atcgcagttc acagtcagac gatccagatt 4740

ccccattgtc ctcagggatg ggattctctg tggattggtt attccttcat gatgcataca 4800

agtgcagggg cagaaggctc aggtcaagcc ctagcctccc ctggttcctg cttggaagag 4860

tttcgttcag ctcccttcat cgaatgtcat gggaggggta cctgtaacta ctatgccaac 4920

tcctacagct tttggctggc aactgtagat gtgtcagaca tgttcagtaa acctcagtca 4980

gaaacgctga aagcaggaga cttgaggaca cgaattagcc gatgtcaagt gtgcatgaag 5040

aggacataa 5049 (SEQ ID NO：3)，

The protein of its coding has aminoacid sequence as follows:

MKLRGVSLAA GLFLLALSLW GQPAEAAACY GCSPGSKCDC SGIKGEKGER GFPGLEGHPG 60

LPGFPGPEGP PGPRGQKGDD GIPGPPGPKG IRGPPGLPGF PGTPGLPGMP GHDGAPGPQG 120

IPGCNGTKGE RGFPGSPGFP GLQGPPGPPG IPGMKGEPGS IIMSSLPGPK GNPGYPGPPG 180

IQGLPGPTGI PGPIGPPGPP GLMGPPGPPG LPGPKGNMGL NFQGPKGEKG EQGLQGPPGP 240

PGQISEQKRP IDVEFQKGDQ GLPGDRGPPG PPGIRGPPGP PGGEKGEKGE QGEPGKRGKP 300

GKDGENGQPG IPGLPGDPGY PGEPGRDGEK GQKGDTGPPG PPGLVIPRPG TGITIGEKGN 360

IGLPGLPGEK GERGFPGIQG PPGLPGPPGA AVMGPPGPPG FPGERGQKGD EGPPGISIPG 420

PPGLDGQPGA PGLPGPPGPA GPHIPPSDEI CEPGPPGSPG DKGLQGEQGV KGDKGDTCFN 480

CIGTGISGPP GQPGLPGLPG PPGSLGFPGQ KGEKGQAGAT GPKGLPGIPG APGAPGFPGS 540

KGEPGDILTF PGMKGDKGEL GSPGAPGLPG LPGTPGQDGL PGLPGPKGEP GGITFKGERG 600

PPGNPGLPGL PGNIGPMGPP GFGPPGPVGE KGIQGVAGNP GQPGIPGPKG DPGQTITQPG 660

KPGLPGNPGR DGDVGLPGDP GLPGQPGLPG IPGSKGEPGI PGIGLPGPPG PKGFPGIPGP 720

PGAPGTPGRI GLEGPPGPPG FPGPKGEPGF ALPGPPGPPG LPGFKGALGP KGDRGFPGPP 780

GPPGRTGLDG LPGPKGDVGP NGQPGPMGPP GLPGIGVQGP PGPPGIPGPI GQPGLHGIPG 840

EKGDPGPPGL DVPGPPGERG SPGIPGAPGP IGPPGSPGLP GKAGASGFPG TKGEMGMMGP 900

PGPPGPLGIP GRSGVPGLKG DDGLQGQPGL PGPTGEKGSK GEPGLPGPPG PMDPNLLGSK 960

GEKGEPGLPG IPGVSGPKGY QGLPGDPGQP GLSGQPGLPG PPGPKGNPGL PGQPGLIGPP 1020

GLKGTIGDMG FPGPQGVEGP PGPSGVPGQP GSPGLPGQKG DKGDPGISSI GLPGLPGPKG 1080

EPGLPGYPGN PGIKGSVGDP GLPGLPGTPG AKGQPGLPGF PGTPGPPGPK GISGPPGNPG 1140

LPGEPGPVGG GGHPGQPGPP GEKGKPGQDG IPGPAGQKGE PGQPGFGNPG PPGLPGLSGQ 1200

KGDGGLPGIP GNPGLPGPKG EPGFHGFPGV QGPPGPPGSP GPALEGPKGN PGPQGPPGRP 1260

GLPGPEGPPG LPGNGGIKGE KGNPGQPGLP GLPGLKGDQG PPGLQGNPGR PGLNGMKGDP 1320

GLPGVPGFPG MKGPSGVPGS AGPEGEPGLI GPPGPPGLPG PSGQSIIIKG DAGPPGIPGQ 1380

PGLKGLPGPQ GPQGLPGPTG PPGDPGRNGL PGFDGAGGRK GDPGLPGQPG TRGLDGPPGP 1440

DGLQGPPGPP GTSSVAHGFL ITRHSQTTDA PQCPQGTLQV YEGFSLLYVQ GNKRAHGQDL 1500

GTAGSCLRRF STMPFMFCNI NNVCNFASRN DYSYWLSTPE PMPMSMQPLK GQSIQPFISR 1560

CAVCEAPAVV IAVHSQTIQI PHCPQGWDSL WIGYSFMMHT SAGAEGSGQA LASPGSCLEE 1620

FRSAPFIECH GRGTCNYYAN SYSFWLATVD VSDMFSKPQS ETLKAGDLRT RISRCQVCMK 1680

RT 1682 (SEQ ID NO：4)。

The COL4A5 gene mutation body that contriver finds is compared with SEQ ID NO:1, have and c.1365_1373delTCCAGGCCC suddenly change, namely relative to wild-type COL4A5 gene, the cDNA of COL4A5 gene mutation body of the present invention lacks the 1365th to the 1373rd bit base TCCAGGCCC.Thus, product coded by it is compared with the COL4A5 of wild-type, have and p.456_458delPGP suddenly change, namely this sudden change causes owing to c.1365_1373delTCCAGGCCC suddenling change, particularly, relative to wild-type COL4A5, isolated polypeptide of the present invention (i.e. COL4A5 mutant) lacks the 456th to the 458th amino acids PGP.

Known at present, one of subunit in COL4A5 genes encoding IV collagen type six subunits, and collagen protein is the important component part of basilar membrane.Wild-type COL4A5 albumen has 1685 amino acid, 161044 Da.Sudden change on COL4A5 gene, can cause the chain AS syndrome of X-.But have not yet to see the relevant report c.1365_1373delTCCAGGCCC sporting the pathogenic mutation of AS syndromes of COL4A5 gene.

According to a second aspect of the invention, the present invention proposes a kind of isolated polypeptide.According to embodiments of the invention, compared with wild-type OL4A5, p.456_458delPGP, this isolated polypeptide has suddenlys change, namely this sudden change causes owing to c.1365_1373delTCCAGGCCC suddenling change, particularly, relative to wild-type COL4A5, isolated polypeptide of the present invention (i.e. COL4A5 mutant) lacks the 456th to the 458th amino acids PGP.According to concrete examples more of the present invention, this polypeptide is by the nucleic acid encoding of the coding COL4A5 mutant of aforementioned separation.By detecting in biological sample whether express this polypeptide, effectively can detect biological sample and whether easily suffering from AS syndrome, also whether can exist in organism by detecting these polypeptide, effectively can predict whether organism easily suffers from AS syndrome.

According to a third aspect of the invention we, the present invention proposes a kind of method of screening the easily syndromic biological sample of trouble AS.According to embodiments of the invention, the method comprises the following steps:

From described extraction from biological material sample of nucleic acid.According to embodiments of the invention, the type of biological sample is also not particularly limited, as long as can extract the sample of nucleic acid whether reflection biological sample COL4A5 exists sudden change from this biological sample.According to embodiments of the invention, biological sample can for being selected from blood of human body, skin, hypodermic at least one, preferred peripheral blood.Thus, can carry out easily sampling and detecting, thus the easy efficiency suffering from the syndromic biological sample of AS of screening can be improved further.According to embodiments of the invention, here used term " sample of nucleic acid " should be interpreted broadly, it can be anyly can reflect in biological sample, whether COL4A5 exists the sample of sudden change, it can be such as the complete genome DNA of extracting directly from biological sample, also can be the part comprising COL4A5 encoding sequence in this full-length genome, can be the total serum IgE extracted from biological sample, also can be the mRNA extracted from biological sample.According to one embodiment of present invention, described sample of nucleic acid is complete genome DNA.Thus, what can expand biological sample carrys out source range, and can determine the much information of biological sample simultaneously, thus can improve the easy efficiency suffering from the syndromic biological sample of AS of screening.In addition, according to embodiments of the invention, for employing RNA as sample of nucleic acid, may further include from extraction from biological material sample of nucleic acid: from extraction from biological material RNA sample, preferred RNA sample is mRNA; And based on obtained RNA sample, by reverse transcription reaction, obtain cDNA sample, the cDNA composition of sample sample of nucleic acid obtained.Thus, the efficiency utilizing RNA as the syndromic biological sample of sample of nucleic acid screening easy trouble AS can be improved further.

Next, after obtaining sample of nucleic acid, can analyze sample of nucleic acid, thus the nucleotide sequence of obtained sample of nucleic acid can be determined.According to embodiments of the invention, determine the method and apparatus of the nucleotide sequence of obtained sample of nucleic acid and be not particularly limited.According to a particular embodiment of the invention, sequence measurement can be passed through, the nucleotide sequence of definite kernel acid sample.According to embodiments of the invention, may be used for the method and apparatus that carries out checking order and be not particularly limited.According to embodiments of the invention, s-generation sequencing technologies can be adopted, also can adopt the third generation and forth generation or more advanced sequencing technologies.According to concrete example of the present invention, can utilize be selected from Hiseq2000, SOLiD, 454 and at least one of single-molecule sequencing device nucleotide sequence is checked order.Thus, in conjunction with up-to-date sequencing technologies, can reach the higher order-checking degree of depth for Single locus, detection sensitivity and accuracy improve greatly, thus can utilize the feature that the high-throughput of these sequencing devices, the degree of depth check order, improve further and carry out detecting the efficiency analyzed to sample of nucleic acid.Thus, follow-up accuracy when sequencing data is analyzed and accuracy can be improved.Thus, according to embodiments of the invention, the nucleotide sequence of definite kernel acid sample may further include: first, for obtained sample of nucleic acid, builds nucleic acid sequencing library; And checked order in obtained nucleic acid sequencing library, to obtain the sequencing result be made up of multiple sequencing data.According to some embodiments of the present invention, can adopt be selected from Hiseq2000, SOLiD, 454 and at least one of single-molecule sequencing device checked order in obtained nucleic acid sequencing library.In addition, according to embodiments of the invention, can screen sample of nucleic acid, enrichment COL4A5 exon, this screening enrichment before structure sequencing library, can build in sequencing library process, or carries out after building sequencing library.According to one embodiment of present invention, for sample of nucleic acid, build nucleic acid sequencing library and comprise further: utilize COL4A5 exon Auele Specific Primer, pcr amplification is carried out to sample of nucleic acid; And for obtained amplified production, build nucleic acid sequencing library.Thus, can pcr amplification be passed through, enrichment COL4A5 exon, thus the easy efficiency suffering from the syndromic biological sample of AS of screening can be improved further.According to embodiments of the invention, the sequence of COL4A5 exon Auele Specific Primer is not particularly limited, and according to a preferred embodiment of the invention, this COL4A5 exon Auele Specific Primer has the nucleotide sequence shown in SEQ ID NO:5 and 6:

Forward primer: 5 '-CATTCCTGGACCTCCTGGACTTGA-3 ' (SEQ ID NO:5);

Reverse primer: 5 '-AGTCAGTGGCTAAGGTGTGATGGA-3 ' (SEQ ID NO:6).

Contriver is surprised to find, and by adopting above-mentioned primer, significantly effectively can complete the amplification to COL4A5 exon in PCR reaction system.It should be noted that, the nucleotide sequence shown in these SEQ ID NO:5 and SEQ ID NO:6 be the present inventor after having paid arduous labor, unexpected to obtain.

About for sample of nucleic acid; build method and the flow process of sequencing library; those skilled in the art suitably can select according to different sequencing technologies; about the details of flow process; the code that can provide see the such as Illumina company of manufacturer of order-checking instrument, for example, see Illumina company Multiplexing Sample Preparation Guide(Part#1005361; Or Paired-End SamplePrep Guide(Part#1005063 Feb2010); Feb2010), by referring to being incorporated to herein.According to embodiments of the invention, from the method and apparatus of extraction from biological material sample of nucleic acid, be also not particularly limited, commercial nucleic acid extraction kit can be adopted to carry out.

It should be noted that, term " nucleotide sequence " used here should make broad understanding, it can be after the sequencing data that obtains of checking order to sample of nucleic acid is assembled, the complete nucleic acid sequence information obtained, also can be directly adopt sequencing data (reads) by checking order obtained to sample of nucleic acid as nucleotide sequence, as long as the encoding sequence containing corresponding COL4A5 in these nucleotide sequences.

Finally, after the nucleotide sequence of definite kernel acid sample, the sequence of the nucleotide sequence of obtained sample of nucleic acid and SEQ ID NO:1 is compared.C.1365_1373delTCCAGGCCC suddenly change if had in obtained nucleotide sequence, then AS syndrome easily suffered from by indicator organism sample.Thus, by easily suffering from the method for the syndromic biological sample of AS according to the screening of the embodiment of the present invention, effectively can screen and easily suffer from the syndromic biological sample of AS.According to embodiments of the invention, the method and apparatus of compare to nucleotide sequence and SEQID NO:1 being also not particularly limited, and the software of any conventional can be adopted to operate, and according to specific examples of the present invention, SOAP software can be adopted to compare.

It should be noted that, the purposes according to " method of the syndromic biological sample of AS is easily suffered from screening " of the embodiment of the present invention is not particularly limited, such as, can be used as the screening method of non-diagnostic object.

System and the test kit of the syndromic biological sample of AS are easily suffered from screening

According to a forth aspect of the invention, the present invention proposes a kind of system effectively can implemented above-mentioned screening and easily suffer from the method for the syndromic biological sample of AS.

With reference to figure 1, according to embodiments of the invention, the system 1000 that the syndromic biological sample of AS is easily suffered from this screening comprises nucleic acid-extracting apparatus 100, nucleotide sequence determining device 200 and judgment means 300.

According to embodiments of the invention, nucleic acid-extracting apparatus 100 is for from extraction from biological material sample of nucleic acid.As previously mentioned, according to embodiments of the invention, the type of sample of nucleic acid is also not particularly limited, and for employing RNA as sample of nucleic acid, then nucleic acid-extracting apparatus comprises RNA extraction unit 101 and reverse transcription unit 102 further, wherein, extraction unit 101 is for from extraction from biological material RNA sample, and reverse transcription unit 102 is connected with RNA extraction unit 101, for carrying out reverse transcription reaction to RNA sample, to obtain cDNA sample, the cDNA composition of sample sample of nucleic acid obtained.

According to embodiments of the invention, nucleotide sequence determining device 200 is connected with nucleic acid-extracting apparatus 100, for analyzing sample of nucleic acid, so that the nucleotide sequence of definite kernel acid sample.As previously shown, the nucleotide sequence of the method definite kernel acid sample of order-checking can be adopted.Thus, according to one embodiment of present invention, described nucleotide sequence determining device 200 may further include: library construction unit 201 and order-checking unit 202.Library construction unit 201, for for sample of nucleic acid, builds nucleic acid sequencing library; Order-checking unit 202 is connected with library construction unit 201, for checking order to nucleic acid sequencing library, to obtain the sequencing result be made up of multiple sequencing data.As previously mentioned, can pcr amplification be passed through, enrichment COL4A5 exon, improve the efficiency that the syndromic biological sample of AS is easily suffered from screening further.Thus, library construction unit 201 may further include pcr amplification module (not shown), COL4A5 exon Auele Specific Primer is provided with in this pcr amplification module, to utilize COL4A5 exon Auele Specific Primer, pcr amplification is carried out to described sample of nucleic acid, according to a particular embodiment of the invention, COL4A5 exon Auele Specific Primer has the nucleotide sequence as shown in SEQ ID NO:5 and 6.According to embodiments of the invention, order-checking unit 202 can comprise and is selected from HISEQ2000, SOLiD, 454 and at least one of single-molecule sequencing device.Thus, in conjunction with up-to-date sequencing technologies, can reach the higher order-checking degree of depth for Single locus, detection sensitivity and accuracy improve greatly, thus can utilize the feature that the high-throughput of these sequencing devices, the degree of depth check order, improve further and carry out detecting the efficiency analyzed to sample of nucleic acid.Thus, improve follow-up accuracy when sequencing data is analyzed and accuracy.

According to embodiments of the invention, judgment means 300 is connected with nucleotide sequence determining device 200, be suitable for the nucleotide sequence of sample of nucleic acid to compare, to judge whether biological sample easily suffers from AS syndrome based on the nucleotide sequence of sample of nucleic acid and the difference of SEQ ID NO:1.Particularly, based on the nucleotide sequence of sample of nucleic acid compared with SEQ ID NO:1, whether have and c.1365_1373delTCCAGGCCC suddenly change, judge whether biological sample easily suffers from AS syndrome.As previously mentioned, according to one embodiment of present invention, the nucleotide sequence of sample of nucleic acid, compared with SEQ ID NO:1, has and c.1365_1373delTCCAGGCCC suddenlys change, and is that biological sample easily suffers from the syndromic instruction of AS.As previously mentioned, according to embodiments of the invention, the equipment of compare to nucleotide sequence and SEQ ID NO:1 being also not particularly limited, and the software of any conventional can be adopted to operate, according to specific examples of the present invention, SOAP software can be adopted to compare.

Thus, utilize this system, effectively can implement the method that the syndromic biological sample of AS is easily suffered from aforementioned screening, thus effectively can screen the easily syndromic biological sample of trouble AS.

According to a fifth aspect of the invention, the present invention proposes a kind of test kit for screening the easily syndromic biological sample of trouble AS.According to embodiments of the invention, this is used for screening the test kit of easily suffering from the syndromic biological sample of AS and comprises: the reagent being suitable for detecting COL4A5 gene mutation body, wherein compared with SEQ ID NO:1, this COL4A5 gene mutation body has and c.1365_1373delTCCAGGCCC suddenlys change.Utilize test kit according to an embodiment of the invention, effectively can screen and easily suffer from the syndromic biological sample of AS.In this article, the term used " is suitable for detecting the reagent of COL4A5 gene mutation body " and should be interpreted broadly, namely can be the reagent detecting COL4A5 encoding gene, also can be the reagent detecting COL4A5 mutant polypeptide, such as, can adopt the antibody in identification specificity site.According to one embodiment of present invention, described reagent is nucleic acid probe or primer, and preferably, described nucleic acid probe or primer have the nucleotide sequence as shown in SEQ ID NO:5-6.Thus, the easily syndromic biological sample of trouble AS can be screened efficiently.

It should be noted that, before this paper, the feature and advantage described in method part of the syndromic biological sample of AS are easily suffered from screening, are equally applicable to screen system or the test kit of the easily syndromic biological sample of trouble AS, do not repeat them here.

Construct and reconstitution cell

According to a sixth aspect of the invention, the invention allows for a kind of construct.According to embodiments of the invention, this construct comprises the nucleic acid of the coding COL4A5 mutant of foregoing separation, i.e. COL4A5 gene mutation body of the present invention.Thus, utilize the reconstitution cell that construct transformed acceptor cell of the present invention obtains, can effectively for screening the syndromic medicine for the treatment of AS.Wherein, the kind of described recipient cell is not particularly limited, and can be such as Bacillus coli cells, mammalian cell, preferred this receptor cell derived be in Mammals.

Term " construct " used in the present invention refers to so a kind of Genetic carrier, and it comprises specific nucleic acid sequence, and can proceed in host cell by object nucleotide sequence, to obtain reconstitution cell.According to embodiments of the invention, the form of construct is not particularly limited.According to embodiments of the invention, it can be at least one of plasmid, phage, artificial chromosome, clay (Cosmid), virus, preferred plasmid.Plasmid, as Genetic carrier, has simple to operate, can carry the character compared with large fragment, convenient operation and process.The form of plasmid is also not particularly limited, and both can be circular plasmids, also can be linear plasmid, namely can be strand, also can be double-strand.Those skilled in the art can select as required.Term " nucleic acid " used in the present invention can be any polymkeric substance comprising deoxyribonucleotide or ribonucleotide, includes but not limited to through that modify or not modified DNA, RNA, and its length is not by any special restriction.For the construct for building reconstitution cell, preferred described nucleic acid is DNA, because DNA is for RNA, it is more stable, and easy handling.

According to a seventh aspect of the invention, the invention allows for a kind of reconstitution cell.According to embodiments of the invention, this reconstitution cell is obtained by foregoing construct transformed acceptor cell.Thus reconstitution cell of the present invention can COL4A5 gene mutation body entrained by expression construct.According to some embodiments of the present invention, utilize reconstitution cell of the present invention, effectively can screen the syndromic medicine for the treatment of AS.According to embodiments of the invention, the kind of recipient cell is not particularly limited, and can be such as Bacillus coli cells, mammalian cell, preferred described recipient cell derives from non-human mammal.

Below with reference to specific embodiment, the present invention will be described, it should be noted that, these embodiments are only illustrative, and can not be interpreted as limitation of the present invention.

If do not specialize, the conventional means that the technique means adopted in embodiment is well known to those skilled in the art, can carry out with reference to " Molecular Cloning: A Laboratory guide " third edition or related products, the reagent adopted and product be also can business obtain.The various process do not described in detail and method are ordinary methods as known in the art, source, the trade(brand)name of agents useful for same and be necessary to list its moiety person, all indicate when occurring first, identical reagent used if no special instructions thereafter, all identical with the content indicated first.

Embodiment 1 determines AS syndrome pathogenic mutation

1, sample collection

Contriver collects only one China AS syndrome family, and its pedigree chart is shown in Fig. 2.As shown in Figure 2, wherein, represent normal male, represent male patient, represent female patient, arrow indication is propositus, and propositus is identical twin.

Contriver gathers the peripheral blood sample obtaining normal people in patient and family in above-mentioned AS syndrome patient family.

2, order-checking is caught in target area

Contriver utilizes chip NimbleGen Sequence Capture Arrays in conjunction with Illumina Hiseq2000 high throughput sequencing technologies, two in above-mentioned AS syndrome patient family is suffered to the COL4A3(MIM of propositus: 120070), COL4A4(MIM:120131) and the Coding region sequence of COL4A5(MIM:303630) gene check order.

Specific as follows:

2.1DNA extract

Gather the peripheral blood of two propositus (III-1 and III-2) in AS syndrome patient family shown in Fig. 2, utilize the genomic dna in white corpuscle cracking process extracting peripheral blood leucocyte, and utilize concentration and the purity of spectrophotometer measurement DNA, the OD260/OD280 of each genomic dna of gained all should between 1.7-2.0, concentration is no less than 200 nanograms/microlitre, and total amount is no less than 3 micrograms.

2.2 target areas are caught and order-checking

Utilize ultrasonoscope (CovarisS2, Massachusetts, USA) each genomic dna sample is broken at random the fragment of about 200-300bp, subsequently according to the process specifications that manufacturers provides, connect top connection at fragment two ends respectively and prepare library (can be see: the Illumina/Solexa standard that http://www.illumina.com/ provides builds storehouse specification sheets, be incorporated in full herein by referring to by it).Hybridization enrichment is carried out through the linear amplification of Ligation-mediated PCR (LM-PCR) and capture agent Biotinylated DNA Library after library is purified, again through the linear amplification of LM-PCR, namely be available on the machine after library detection is qualified order-checking, to obtain raw sequencing data.Wherein, order-checking platform is Illumina Hiseq2000, and reading length is 90bp, the average order-checking degree of depth of each sample is minimum is 150 ×.

3, variation detection, annotation and database compare

The raw sequencing data of Illumina basecalling Software1.7 to above-mentioned acquisition is utilized to process, after filtering and depolluting, use SOAPaligner/SOAP2(can be see: Li R, Li Y, Kristiansen K, et al, SOAP:shortoligonucleotide alignment program.Bioinformatics2008,24 (5): 713-714; Li R, Yu C, Li Y, ea al, SOAP2:an improved ultrafast tool for short read alignment.Bioinformatics2009,25 (15): 1966-1967, being incorporated in full herein by referring to by it) comparison is to UCSC mankind's reference genome (hg19, build37.1, http://genome.ucsc.edu/), to obtain comparison to the unique aligned sequences on genome.Then utilize SOAPsnp (can be see: Li R, Li Y, Fang X, Yang H, et al, SNP detection for massively parallelwhole-genome resequencing.Genome Res2009,19 (6): 1124-1132, is incorporated in full herein by referring to by it) determine the genotype of target region.

Subsequently by dbSNP database (http://hgdownload.cse.ucsc.edu/goldenPath/hg19/database/snp135. txt.gz.), HapMap database (ftp: //ftp.ncbi.nlm.nih.gov/hapmap), thousand human genome databases (ftp: //ftp.1000genomes.ebi.ac.uk/vol1/ftp), the filtration of the public databases such as Yan Di and Huang Di, two legendary rulers of remote antiquity's database (http://yh.genomics.org.cn/), remove all known and gene frequency variations of being greater than 0.005 in a database in the sequencing result of above-mentioned acquisition, the final nonsynonymous mutation only retained on coding region.

Then, consider sequencing quality and bioinformatic analysis result, only screen one and be positioned at suspicious sudden change on COL4A5 gene c.1365_1373delTCCAGGCCC(p.456_458delPGP), namely on this gene, lack the base of 1365-1373 position, the albumen of this genes encoding is caused to lack to 458 at 456, than 1685 AA of wild-type protein, mutein only has 1682 Aa.

Known, COL4A5 gene is the syndromic Disease-causing gene of AS, one of subunit of COL4A5 coding IV collagen type six subunits, and collagen protein is the important component part of basilar membrane.Sudden change then on COL4A5 gene is the pathogenic mutation causing the chain AS of X-, and above-mentioned c.1365_1373delTCCAGGCCC sudden change is not yet reported at present, is the new mutant of known.

To sum up, contriver thinks, the c.1365_1373delTCCAGGCCC sudden change of COL4A5 gene is very likely the syndromic pathogenic mutation of AS.

Embodiment 2Sanger method sequence verification

Respectively the COL4A5 gene of the family member (I2, II1, II2, II4, III1, III2, III3) in the AS syndrome patient family described in embodiment 1 is detected: for the c.1365_1373delTCCAGGCCC sudden change design primer of COL4A5 gene, then mutational site relevant sequence is obtained by the method for pcr amplification, product purification and order-checking, according to determining that sequencing results belongs to saltant type or wild-type, checking COL4A5 gene and the dependency between this sudden change and AS syndrome.

Concrete grammar step is as follows:

1, DNA extraction

According to the method for the extraction DNA described in embodiment 1, extract the genomic dna in preparation experimenter peripheric venous blood respectively, for subsequent use.

2, design of primers and PCR reaction

First, reference men and women's genoid data unit sequence storehouse GRCh37/hg19, design obtains the COL4A5 gene extron Auele Specific Primer with nucleotide sequence shown in SEQ ID NO:5-6, and concrete sequence is as follows:

Forward primer: 5 '-CATTCCTGGACCTCCTGGACTTGA-3 ' (SEQ ID NO:5);

Reverse primer: 5 '-AGTCAGTGGCTAAGGTGTGATGGA-3 ' (SEQ ID NO:6).

Then, prepare the PCR reaction system of each genomic dna sample according to following proportioning respectively and carry out PCR reaction:

Reaction system:

Reaction system: 20 μ L

PCR reaction conditions:

Thus, the pcr amplification product that each receptor gene organizes DNA sample is obtained.

3, check order

The pcr amplification product that each receptor gene obtained in step 2 organizes DNA sample is directly carried out DNA sequencing.Wherein, order-checking adopts ABI3730 type sequenator to carry out.

Based on sequencing result, in AS patient's family of the present invention, sudden change investigation is carried out to COL4A5 gene, found that c.1365_1373delTCCAGGCCC the male sex family member (I2 and II1) do not fallen ill all does not carry (p.456_458delPGP) sudden change, female patient (II2 and II4) is all with heterozygous mutant, and male patient (III1, III2, III3) all carries (p.456_458delPGP) sudden change of isozygotying c.1365_1373delTCCAGGCCC.Wherein, Fig. 3 shows in above-mentioned AS syndrome patient family, the representative Sanger sequence verification peak figure in the COL4A5 gene of non-affected males (for normal sequence), female patient (tool heterozygous mutant) and male patient's (tool homozygous mutation) c.1365_1373delTCCAGGCCC mutational site.As shown in Figure 3, dotted line frame is the position starting in sudden change to lack, and solid box is the sequence sequentially moved forward after disappearance.

Further, in conjunction with family collection of illustrative plates and patient profiles, and this type of disease X is chain there is the infull situation of women's penetrance, and contriver thinks, this mutational site meets the X hereditary pattern reported, thus family internal evidence understand genotype and phenotype exist be divided into from.Thus, prove that COL4A5 gene is the pathogenic mutation c.1365_1373delTCCAGGCCC sporting this disease of the syndromic Disease-causing gene of AS, COL4A5 gene further.

Embodiment 3

Catch sequence measurement according to above-mentioned target area, namely distribute patient to the patient of 1 outside family and carry out target area and catch sequence verification, result all finds no suspicious pathogenic mutation in its whole COL4A5 gene coding region.

Then, detect the c.1365_1373delTCCAGGCCC sudden change of COL4A5 gene in the agnate irrelevant normal control database of 100 example, result all fails this sudden change to be detected, proves that this mutational site is the sudden change of very low frequency.

Thus, c.1365_1373delTCCAGGCCC (p.456_458delPGP) sudden change further demonstrating in embodiment 1 COL4A5 gene in this AS family detecting acquisition is the pathogenic mutation of AS syndromes.

Embodiment 4 detection kit

Prepare a detection kit, it comprises the primer c.1365_1373delTCCAGGCCC suddenlyd change that can detect COL4A5 gene, easily the syndromic biological sample of AS is suffered from for screening, wherein these primers are COL4A5 gene extron Auele Specific Primer, and its sequence is as described in example 1 above shown in SEQ ID NO:5-6.

Utilize mentioned reagent box to screen the concrete steps easily suffering from the syndromic biological sample of AS to be: according to embodiment 2 step 1 described in method extract person DNA to be measured, with extracted DNA for the exon Auele Specific Primer of template and above-mentioned COL4A5 gene carries out PCR reaction, and according to this area ordinary method to PCR primer purifying, the product of purifying is checked order, c.1365_1373delTCCAGGCCC, whether the sequence then obtained by observing order-checking has is suddenlyd change, effectively can detect COL4A5 gene mutation body of the present invention whether to exist in person DNA to be measured, thus effectively can detect person to be measured whether easily trouble AS syndrome, further, can filter out from person to be measured and easily suffer from the syndromic biological sample of AS.

In the description of this specification sheets, specific features, structure, material or feature that the description of reference term " embodiment ", " some embodiments ", " example ", " concrete example " or " some examples " etc. means to describe in conjunction with this embodiment or example are contained at least one embodiment of the present invention or example.In this manual, identical embodiment or example are not necessarily referred to the schematic representation of above-mentioned term.And the specific features of description, structure, material or feature can combine in an appropriate manner in any one or more embodiment or example.

Although illustrate and describe embodiments of the invention, those having ordinary skill in the art will appreciate that: can carry out multiple change, amendment, replacement and modification to these embodiments when not departing from principle of the present invention and aim, scope of the present invention is by claim and equivalents thereof.

Claims (10)

1. a nucleic acid for the coding COL4A5 mutant be separated, it is characterized in that, described nucleic acid, compared with SEQ ID NO:1, has and c.1365_1373delTCCAGGCCC suddenlys change,

Optionally, described nucleic acid is DNA.

2. an isolated polypeptide, is characterized in that, compared with SEQ ID NO:2, p.456_458delPGP described isolated polypeptide has suddenlys change,

Optionally, described polypeptide is by nucleic acid encoding according to claim 1.

3. a method for the syndromic biological sample of AS is easily suffered from screening, it is characterized in that, comprises the following steps:

From described extraction from biological material sample of nucleic acid;

Determine the nucleotide sequence of described sample of nucleic acid;

The nucleotide sequence of described sample of nucleic acid is compared with SEQ ID NO:1, and having c.1365_1373delTCCAGGCCC sudden change is that described biological sample easily suffers from the syndromic instruction of AS,

Optionally, described biological specimen for being selected from blood of human body, skin, hypodermic at least one,

Optionally, described sample of nucleic acid is complete genome DNA.

4. method according to claim 3, is characterized in that, comprises further from described extraction from biological material sample of nucleic acid:

From described extraction from biological material RNA sample, preferred described RNA sample is mRNA; And

Based on described RNA sample, by reverse transcription reaction, obtain cDNA sample, sample of nucleic acid described in described cDNA composition of sample.

5. method according to claim 3, is characterized in that, determines that the nucleotide sequence of described sample of nucleic acid comprises further:

For described sample of nucleic acid, build nucleic acid sequencing library; And

Checked order in described nucleic acid sequencing library, to obtain the sequencing result be made up of multiple sequencing data, optionally, adopt be selected from Hiseq2000, SOLiD, 454 and at least one of single-molecule sequencing device checked order in described nucleic acid sequencing library,

Optionally, for described sample of nucleic acid, build nucleic acid sequencing library and comprise further:

Utilize COL4A5 gene extron Auele Specific Primer, pcr amplification is carried out to described sample of nucleic acid; And

For obtained amplified production, build described nucleic acid sequencing library,

Optionally, described Auele Specific Primer has the nucleotide sequence as shown in SEQ ID NO:5-6.

6. a system for the syndromic biological sample of AS is easily suffered from screening, it is characterized in that, comprising:

Nucleic acid-extracting apparatus, described nucleic acid-extracting apparatus is used for from described extraction from biological material sample of nucleic acid;

Nucleotide sequence determining device, described nucleotide sequence determining device is connected with described nucleic acid-extracting apparatus, for analyzing described sample of nucleic acid, to determine the nucleotide sequence of described sample of nucleic acid;

Judgment means, whether described judgment means is connected with described nucleotide sequence determining device, based on the nucleotide sequence of described sample of nucleic acid compared with SEQ ID NO:1, to have and c.1365_1373delTCCAGGCCC to suddenly change, judge whether described biological sample easily suffers from AS syndrome

Optionally, described nucleic acid-extracting apparatus comprises further:

RNA extraction unit, described RNA extraction unit is used for from described extraction from biological material RNA sample; And

Reverse transcription unit, described reverse transcription unit is connected with described RNA extraction unit, for carrying out reverse transcription reaction to described RNA sample, to obtain cDNA sample, sample of nucleic acid described in described cDNA composition of sample.

7. system according to claim 6, is characterized in that, described nucleotide sequence determining device comprises further:

Library construction unit, described library construction unit is used for for described sample of nucleic acid, builds nucleic acid sequencing library; And

Order-checking unit, described order-checking unit is connected with described library construction unit, for checking order to described nucleic acid sequencing library, to obtain the sequencing result be made up of multiple sequencing data,

Optionally, described library construction unit comprises further:

Pcr amplification module, is provided with COL4A5 gene extron Auele Specific Primer in described pcr amplification module, to utilize described Auele Specific Primer, carry out pcr amplification to described sample of nucleic acid,

Optionally, described Auele Specific Primer has the nucleotide sequence as shown in SEQ ID NO:5-6,

Optionally, described order-checking unit comprises and is selected from HISEQ2000, SOLiD, 454 and at least one of single-molecule sequencing device.

8., for screening a test kit of easily suffering from the syndromic biological sample of AS, it is characterized in that, contain:

Be suitable for the reagent detecting COL4A5 gene mutation body, wherein compared with SEQ ID NO:1, described COL4A5 gene mutation body has and c.1365_1373delTCCAGGCCC suddenlys change,

Optionally, described reagent is nucleic acid probe or primer,

Optionally, described nucleic acid probe or primer have the nucleotide sequence as shown in SEQ ID NO:5-6.

9. a construct, is characterized in that, comprises the nucleic acid of the coding COL4A5 mutant of separation according to claim 1.

10. a reconstitution cell, is characterized in that, described reconstitution cell is obtained by construct transformed acceptor cell according to claim 9.